To further understand the genotype-dependent drought response mechanisms in tobacco (Nicotiana tabacum L.), an integrated physio-biochemical and molecular analysis was performed on four tobacco cultivars with different drought tolerance (Y6, Y10, Y87, and Hongda). Our data showed that PEG-induced dehydration significantly affected plant growth expressed as biomass accumulation, leaf gas-exchange, cellular redox homeostasis, and gene expression. Compared to Y87 and Hongda, Y6 and Y10 exhibited less water loss, delayed leaf senescence, better chloroplast integrity, and higher photosynthetic activity under stressed conditions, suggesting that Y6 and Y10 were more drought-tolerant than Y87 and Hongda. Biochemical analysis has clearly highlighted that the plant's tolerance to dehydration is related to the control of ROS generation and lipid peroxidation, supported by higher levels of antioxidant defenses detected in Y6 and Y10. At the molecular level, the expression of stress-associated genes was higher in Y6 and Y10 during drying than in Y87 and Hongda, suggesting that higher expression of representative defense genes might be a more effective response to mitigate dehydration in tobacco.